The long-term goal of this application is to unravel mechanisms underlying the precise molecular controls of bacterial cell division. The project described here, centered on the FtsZ protein, is the first step toward this goal. FtsZ is highly conserved across bacterial species and has been identified as a novel drug target for antimicrobial therapy. In vivo FtsZ assembles into a supramolecular ring-like structure (hence named the Z-ring) at the leading edge of the invaginating membrane to drive cell division. The objective of this project is to determine the structure of the Z-ring, in particular the arrangement of protofilaments inside the Z-ring (Aim 1), and to identify the contraction mechanism of the Z-ring (Aim 2). In Aim 1, using a single-molecule based superresolution imaging technique the Z-ring was imaged with a 30-nm spatial resolution. A 3D bundle arrangement of FtsZ protofilaments instead of a 2D flat ribbon was proposed. Such an arrangement model will be further verified by examining three predictions stemmed from the model. The final product of this aim is a high resolution Z-ring structural model that will be instrumental in understanding the contraction mechanism of the Z-ring. In Aim 2, two competing Z-ring contraction mechanisms-disassembly vs. condensation-will be examined. The contraction speed of the Z-ring during the constriction period will be measured using the superresolution imaging technique. This speed will then be compared with the calculated speed based on single-molecule measurements of the disassembly rate and density change of the Z-ring. The role of GTP hydrolysis in Z-ring contraction will also be examined using an FtsZ GTPase mutant. These experiments, combined with the structural model that will be established in Aim 1, will provide important insight into the molecular mechanism of Z-ring contraction. Successful completion of the proposed work will provide: (1) a high-resolution Z- ring structural model; (2) a mechanism of how this structure contracts and (3) a set of innovative in vivo single-molecule assays.